The long range goals of this research project are to understand the role played by paramagnetic centers in the catalytic mechanism of several enzymes, to develop correlations between the detailed electronic structures, gained using advanced Electron Paramagnetic Resonance (EPR) methods, and chemical reactivity, and to further the development of advanced EPR methods and data analysis strategies for solving problems in biological systems. The enzymes to be studied are two multicopper oxidases, FET3p and R. vernicifera laccase, and two alpha-ketoglutarate dependent non-heme iron dioxygenases, taurine dioxygenase and AIkB. All of these proteins catalyze reactions that involve paramagnetic intermediate states at some point in their mechanistic cycle. The experiments proposed in this grant will use EPR spectroscopy at both 9 and 95 GHz. Conventional, continuous wave studies will be used along with the "advanced" EPR techniques of cw- and spin echo detected-ENDOR, and ESEEM in 1- and 2-dimensions. These advanced technologies can be used at both conventional, X-band frequencies (9 GHz) and at W-band (95 GHz). The specific aims of the proposal focus on characterizing the detailed electronic structures of the metal ions that form the catalytic sites of the metalloenzymes under study. Rapid Freeze Quench methods will be used to trap key catalytic intermediates. The data will provide information on the electronic structure of these metals, the coordination of ligands and how both electronic structure and ligand bonding change during catalytic turnover. EPR methods are uniquely suited to obtain this information for paramagnetic centers. Of the four enzymes to be studied, two are directly tied to health related issues. The AIkB protein has recently been identified as having the ability to catalyze the repair of methylated adenine and cytosine bases in both single- and double-stranded DNA. The Fet3p enzyme is a multicopper oxidase that shows ferroxidase activity and thus, shares a common reactivity with ceruloplasrnin, a mammalian rnulticopper ferroxidase that is vital to metal ion homeostasis.